Medium for inducing sporulation of pythium tropicale, preparation method and application thereof

Abstract

The present application relates to the field of microorganism, especially to the culture medium for inducing sporulation of Pythium tropicale, preparation method and application. The culture medium and the method for inducing zoospore production of Pythium tropicale provided by the present application have a zoospore yield of 1.32*10 6 The main raw materials of the present application are cucumber, pepper leaves and vitamin C, which are cheap, and the preparation method is simple and easy to operate. The obtained zoospore suspension is pure and sterile, and is very suitable for the mechanism research and prevention and control technology research and development of Pythium tropicale.

Description

Technical Field

[0001] This invention relates to the field of microbiology, and in particular to a culture medium, preparation method and application for inducing sporulation of Phytophthora tropicalis. Background Technology

[0002] Pepper (Piper nigrum Linn.) is a highly valuable food and spice crop, widely cultivated in tropical countries worldwide. In China, the planting area of ​​pepper reaches over 400,000 mu (approximately 26,667 hectares), with an annual output value exceeding 3 billion yuan, making it a vital tropical crop industry affecting the livelihoods of over one million farmers. Pepper wilt is extremely destructive and contagious, making it the leading disease affecting pepper production globally. In the 1970s, this disease led to a 20% reduction in pepper planting area. In recent years, pepper wilt has become widespread and severely damaging in all pepper-growing areas of Hainan Province, showing a trend of resurgence and increasing severity, becoming one of the key factors affecting pepper production. Pepper wilt is mainly spread through zoospores via rain splash, field runoff, and agricultural operations. Controlling the source of infection and interrupting transmission routes are consistent strategies for controlling this disease. Understanding zoospores is key to revealing the pathogenic mechanism of pepper wilt and is a focus of attention for scientists both domestically and internationally.

[0003] The pathogen causing pepper blight was previously identified as *Phytophthora capsici*. However, studies have shown that some strains isolated from tropical crops such as pepper exhibit significant biological and genetic differences compared to strains isolated from temperate hosts, leading to their separation from *Phytophthora capsici* and naming them *P. tropicalalis*. Many sporulation media for *P. capsici* have been reported in China, including V8 vegetable juice medium (V8), rye medium (RA), carrot medium (CA), soybean medium (BA), orange juice medium (OA), oat medium (OMA), and corn flour medium (CMA), among others. However, these media and methods have low or even no sporulation efficiency in inducing sporulation in *P. tropicalalis*. Currently, there are no highly efficient media and methods for inducing zoospores in *P. tropicalalis*, which is highly detrimental to research on the pathogenic mechanisms of diseases affecting tropical crops such as pepper and the development of control technologies. Summary of the Invention

[0004] Although *Phytophthora tropicalis* is biologically and genetically very similar to *Phytophthora capsici*, the zoospore induction medium for *Phytophthora capsici* is not very efficient on *Phytophthora tropicalis*. This invention provides a simple and efficient culture medium for inducing zoospores of *Phytophthora tropicalis* and its application method to meet the needs of relevant laboratory molecular mechanism research and disease monitoring and control in production.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] In a first aspect, the present invention provides a culture medium for inducing the production of zoospores by Phytophthora tropicalis, comprising cucumber, pepper leaves and vitamin C.

[0007] In some specific embodiments of the present invention, the culture medium comprises the following components, in parts by weight:

[0008] 150-250 portions of cucumber

[0009] 20-30 parts by weight of pepper leaves

[0010] Vitamin C 0.00001 to 0.00002 parts by weight.

[0011] In some specific embodiments of the present invention, the culture medium comprises the following components, in parts by weight:

[0012] 150 servings of cucumber

[0013] 20 parts by weight of pepper leaves

[0014] Vitamin C 0.00002 parts by weight

[0015] or

[0016] 200 servings of cucumber

[0017] 30 parts by weight of pepper leaves

[0018] Vitamin C 0.000015 parts by weight

[0019] or

[0020] 250 servings of cucumber

[0021] 25 parts by weight of pepper leaves

[0022] Vitamin C 0.00002 parts by weight

[0023] or

[0024] 200 servings of cucumber

[0025] 30 parts by weight of pepper leaves

[0026] Vitamin C 0.00001 parts by weight.

[0027] In some specific embodiments of the present invention, the culture medium further includes 15 to 20 parts by weight of agar powder.

[0028] Secondly, the present invention also provides a method for preparing the culture medium, wherein the cucumber is used by crushing the cucumber, filtering it through double-layer gauze to remove residue, and collecting the filtrate; and / or

[0029] The method of using the pepper leaves is to crush the pepper leaves, filter them through double-layered gauze to remove the residue, and collect the filtrate; and / or

[0030] The method of using the vitamin C is to prepare a 1000-fold stock solution of the vitamin C and store it at 4°C. When using, add it to the culture medium solution cooled to 50°C.

[0031] In some specific embodiments of the present invention, the method of using the vitamin C is as follows: dissolve 0.5-1g of the vitamin C in 50mL of 1% oxalic acid solution, filter and store at 4°C to obtain a vitamin C additive; when using, add 0.1% of the vitamin C additive to a culture medium at 50°C.

[0032] In some specific embodiments of the present invention, the preparation method includes:

[0033] Take 150-250g of the cucumber, crush it, and then filter it.

[0034] Take 20-30g of pepper leaves, crush them, collect the filtrate, add 15-20g of agar powder to the filtrate, add deionized water to make up to 1L, and sterilize at 121℃ for 20min.

[0035] Dissolve 0.5-1g of the vitamin C in 50mL of 1% oxalic acid solution, filter, and refrigerate at 4°C to obtain the vitamin C additive. When using, melt the culture medium and cool it to 50°C, add 0.1% of the vitamin C additive, mix well, pour into a sterile petri dish, and solidify to obtain the culture medium plate.

[0036] In some specific embodiments of the present invention, the preparation method includes:

[0037] Take 150-250g of fresh, prickly cucumber, crush it in a food processor, and filter it through double-layered gauze. Similarly, crush 20-30g of fresh pepper leaves and collect the filtrate. Add 15-20g of agar powder to the filtrate, and dilute to 1L with deionized water. Sterilize at 115-121℃ for 15-20 minutes. Dissolve 0.5-1g of vitamin C in 50mL of 1% oxalic acid solution, filter through a sterile filter with a 0.22μm pore size, and store separately at 4℃ as an additive. When using, thaw the culture medium and cool it to about 50℃, add 0.1% by volume of the vitamin C additive, mix well, pour into sterile petri dishes, and allow it to solidify to form the culture medium plates.

[0038] Thirdly, the present invention also provides the application of the culture medium prepared by the method of the culture medium in inducing zoospores of Phytophthora tropicalis.

[0039] Fourthly, the present invention also provides a method for inducing zoospores of Phytophthora tropicalis, wherein Phytophthora tropicalis is inoculated into the culture medium or the culture medium prepared by the preparation method and cultured.

[0040] In some specific embodiments of the present invention, the induction method includes the following steps:

[0041] Step 1: Inoculate the tropical phytophthora into the culture medium and incubate at 26°C for 4–7 days until the mycelium has covered the entire plate.

[0042] Step 2: Press the mycelium to adhere it to the culture medium, and induce it under light at 25-28℃ and 4000-6000 lx for 24-48 h;

[0043] Step 3: After adding sterile water, refrigerate at 4°C for 45–90 min, then keep at 26°C for 30–60 min, and collect the spore suspension;

[0044] Step 4: Observe and count the number of zoospores.

[0045] In some specific embodiments of the present invention, the induction method includes the following steps:

[0046] Step 1: Inoculate a block of *Phytophthora tropicalis* mycelium, approximately 0.5 cm in diameter, into the center of a culture medium plate and incubate at 26°C under light and biochemical conditions for 4–7 days, until the mycelium has completely covered the plate.

[0047] Step 2: Gently press the mycelia on the plate with a sterile L-shaped glass rod to make them adhere to the culture medium, and induce them under light at 25-28℃ and 4000-6000 lx for 24-48 hours;

[0048] Step 3: Add 10 mL of sterile water to the plate and place it in a 4°C refrigerator for 45–90 min, then keep it at 26°C for 30–60 min. Collect the spore suspension in the plate.

[0049] Step 4: Take a small amount of spore suspension and add it to a hemocytometer. Observe and count the number of zoospores under a microscope.

[0050] In some specific embodiments of the present invention, the induction method includes the following steps:

[0051] Step 1: Inoculate a block of tropical Phytophthora mycelium about 0.5 cm in diameter into the center of a culture medium plate and incubate it in a 26°C light biochemical incubator for 5 days.

[0052] Step 2: Gently press the mycelium on the plate with a sterile L-shaped glass rod to make it adhere to the culture medium, and induce it under light at 26℃ and 5000 lx for 30 h;

[0053] Step 3: Add 10 mL of sterile water to the plate and place it in a 4°C refrigerator for 60 min, then keep it at 26°C for another 60 min. Collect the spore suspension in the plate.

[0054] Step 4: Take a small amount of spore suspension and add it to a hemocytometer. Observe and count the number of zoospores under a microscope.

[0055] The culture medium and method for inducing zoospore production by *Phytophthora tropicalis* provided by this invention achieve a zoospore yield of 1.32 × 10⁻⁶. 6 The concentration of zoospores per ml is significantly higher than that of similar culture media. The main raw materials of this invention are cucumber, pepper leaves, and vitamin C, which are inexpensive and easy to prepare. The resulting zoospore suspension is pure and sterile, making it highly suitable for research on the mechanism of tropical fungi and the development of control technologies. Attached Figure Description

[0056] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0057] Figure 1 The results of sequence alignment analysis of the YptI fragment of *Phytophthora tropicalis* isolated from pepper and *Phytophthora capsici* are shown.

[0058] Figure 2 The number of zoospores induced in Example 3 of the present invention is shown under a microscope at 40x magnification.

[0059] Figure 3 The number of zoospores induced by rye culture medium in Comparative Example 1 is shown under a microscope at 40x magnification. Detailed Implementation

[0060] This invention discloses a culture medium, preparation method, and application for inducing sporulation of *Phytophthora tropicalis*. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired results. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0061] Culture medium and preparation method:

[0062] The culture medium preparation method of this invention is as follows: For the culture medium portion: 150-250g of fresh, prickly cucumber is crushed in a food processor and filtered through double-layered gauze. 20-30g of fresh pepper leaves are similarly crushed, and the filtrate is collected. 15-20g of agar powder is added to the filtrate, and deionized water is added to bring the volume to 1L. The solution is then sterilized at 115-121℃ for 15-20 minutes, preferably at 121℃ for 20 minutes. 0.5-1g of vitamin C is dissolved in 50mL of a 1% oxalic acid solution, filtered through a sterile filter with a 0.22μm pore size, and stored separately at 4℃ as an additive. When using, the culture medium is melted and cooled to approximately 50℃, and 0.1% (by volume) of the vitamin C additive is added. The mixture is then poured into sterile petri dishes, and after solidification, the culture medium plate is obtained.

[0063] Zoospore induction methods:

[0064] 1) Inoculate a block of tropical Phytophthora mycelium about 0.5 cm in diameter into the center of a culture medium plate and incubate it in a 26°C light biochemical incubator for 4-7 days, preferably 5 days.

[0065] 2) Gently press the mycelia on the plate with a sterile L-shaped glass rod to make them adhere to the culture medium. Induce light at 25-28℃ and 4000-6000 lx for 24-48 h, preferably at 26℃ and 5000 lx for 30 h.

[0066] 3) After adding 10 mL of sterile water to the plate, transfer it to a 4°C refrigerator for 45-90 min, and then keep it at 26°C for 30-60 min. Preferably, after adding 10 mL of sterile water to the plate, transfer it to a 4°C refrigerator for 60 min, and then keep it at 26°C for 60 min to collect the spore suspension in the plate.

[0067] 4) Take a small amount of spore suspension and drop it onto a hemocytometer, then observe and count the number of zoospores under a microscope.

[0068] The beneficial effects of the present invention include, but are not limited to:

[0069] The culture medium and method for inducing zoospore production by *Phytophthora tropicalis* provided by this invention achieve a zoospore yield of 1.32 × 10⁻⁶. 6 The concentration of zoospores per ml is significantly higher than that of similar culture media. The main raw materials of this invention are cucumber, pepper leaves, and vitamin C, which are inexpensive and easy to prepare. The resulting zoospore suspension is pure and sterile, making it highly suitable for research on the mechanism of tropical fungi and the development of control technologies.

[0070] The raw materials and reagents used in the culture medium, preparation method and application of Phytophthora tropicalis induced to produce sporulation provided by this invention are all commercially available.

[0071] The standard strain of *Phytophthora tropicalis* has the accession number CBS 434.91 at CBS, and can be purchased directly from their official website.

[0072] CBS (Centraalbureauvoor Schimmelcultures), the Dutch Center for the Preservation of Microbial Cultures, is a semi-governmental major center for the preservation of fungal and yeast cultures.

[0073] The center primarily engages in research on microbial culture preservation methods, taxonomy, molecular biology, and medical microbiology. It holds 35,000 strains of fungi and 5,500 strains of yeast. The microbial cultures preserved at the center are available for sale.

[0074] The present invention will be further illustrated below with reference to the embodiments:

[0075] Example 1

[0076] The pathogen was sampled and purified from pepper plants infected with wilt disease in the fields of Hainan Island. The pathogen was identified by YptI gene sequence alignment analysis. It was found that the strain used in this invention had a 12-base deletion (Gap) compared to the *Phytophthora capsici* strain isolated from pepper plants, indicating that the strain used was *Phytophthora tropicalis*. See Bowers JH, Martin FN, Tooley PW, et al. Genetic and Morphological Diversity of Temperate and Tropical Isolates of *Phytophthora capsici* [J]. *Phytopathology*, 2007, 97(4):492-503. or Jeevalatha A, Biju CN, BhaiR S. Ypt1 gene-based recombine polymerase amplification assay for *Phytophthora capsici* and *P. tropicalis* detection in blackpepper [J]. *European Journal of Plant Pathology*, 2021:1-13. Figure 1 ).

[0077] Example 2

[0078] Culture medium preparation method: Crush 150g of fresh, prickly cucumber in a food processor and filter through double-layered gauze. Crush 20g of fresh pepper leaves in the same way and collect the filtrate. Add 20g of agar powder to the filtrate, and dilute to 1L with deionized water. Sterilize at 121℃ for 20min. Dissolve 1g of vitamin C in 50mL of 1% oxalic acid solution, filter through a sterile filter with a 0.22μm pore size, and store separately at 4℃ as an additive. When using, melt the culture medium and cool to approximately 50℃, add 0.1% (by volume) of the vitamin C additive, mix well, pour into sterile petri dishes, and allow to solidify to form the culture medium plates.

[0079] Zoospore induction methods:

[0080] 1) Inoculate a block of tropical Phytophthora mycelium about 0.5 cm in diameter onto the center of a culture medium plate and incubate it in a 26°C light biochemical incubator for 5 days;

[0081] 2) Gently press the mycelia on the plate with a sterile L-shaped glass rod to make them adhere to the culture medium, and induce them under light at 26℃ and 5000lx for 30h.

[0082] 3) After adding 10 mL of sterile water to the plate, transfer it to a 4°C refrigerator for 1 hour, then keep it at 26°C for another 1 hour, and collect the spore suspension in the plate.

[0083] 4) Take a small amount of spore suspension and drop it onto a hemocytometer, then observe and count the number of zoospores under a microscope.

[0084] Example 3

[0085] Culture medium preparation method: Crush 200g of fresh, prickly cucumber in a food processor and filter through double-layered gauze. Crush 30g of fresh pepper leaves in the same way and collect the filtrate. Add 15g of agar powder to the filtrate, and dilute to 1L with deionized water. Sterilize at 121℃ for 20min. Dissolve 0.75g of vitamin C in 50mL of 1% oxalic acid solution, filter through a sterile filter with a 0.22μm pore size, and store separately at 4℃ as an additive. When using, melt the culture medium and cool to approximately 50℃, add 0.1% (by volume) of the vitamin C additive, mix well, pour into sterile petri dishes, and allow to solidify to form the culture medium plates.

[0086] The method for inducing zoospores is the same as in Example 2.

[0087] Example 4

[0088] Culture medium preparation method: Crush 250g of fresh, prickly cucumber in a food processor and filter through double-layered gauze. Crush 25g of fresh pepper leaves in the same way and collect the filtrate. Add 17.5g of agar powder to the filtrate, and dilute to 1L with deionized water. Sterilize at 121℃ for 20min. Dissolve 1g of vitamin C in 50mL of 1% oxalic acid solution, filter through a sterile filter with a 0.22μm pore size, and store separately at 4℃ as an additive. When using, melt the culture medium and cool to approximately 50℃, add 0.1% (by volume) of the vitamin C additive, mix well, pour into sterile petri dishes, and allow to solidify to form the culture medium plates.

[0089] The method for inducing zoospores is the same as in Example 2.

[0090] Example 5

[0091] Culture medium preparation method: Crush 200g of fresh, prickly cucumber in a food processor and filter through double-layered gauze. Crush 30g of fresh pepper leaves in the same way and collect the filtrate. Add 20g of agar powder to the filtrate, and dilute to 1L with deionized water. Sterilize at 121℃ for 20min. Dissolve 0.5g of vitamin C in 50mL of 1% oxalic acid solution, filter through a sterile filter with a 0.22μm pore size, and store separately at 4℃ as an additive. When using, melt the culture medium and cool to approximately 50℃. Add 0.1% (by volume) of the vitamin C additive, mix well, and pour into sterile petri dishes, 15-20mL / dish. After solidification, the culture medium plate is ready.

[0092] The method for inducing zoospores is the same as in Example 2.

[0093] Comparative Example 1

[0094] Preparation of rye culture medium: Soak 60g of rye seeds in 1000ml of deionized water for 24 hours, autoclave at 121℃ for 20 minutes, and filter through double-layer gauze to remove residue. Add 20g of agar powder and dilute with deionized water to 1000ml. Sterilize at 121℃ for 20 minutes.

[0095] The method for inducing zoospores is the same as in Example 2.

[0096] Comparative Example 2

[0097] Soybean culture medium preparation method: Soak 60g of soybean seeds in deionized water for 24 hours. Remove the soaked soybeans and mix with 300ml of deionized water. Blend in a food processor and filter through double-layered gauze to remove residue. Add 20g of agar powder and dilute with deionized water to 1000ml. Autoclave the culture medium at 121℃ for 20 minutes.

[0098] The method for inducing zoospores is the same as in Example 2.

[0099] Comparative Example 3

[0100] V8 culture medium preparation method: Centrifuge V8 fruit and vegetable juice (brand: Kampo, USA) at 5000g for 5 minutes, then take 100ml of the supernatant, add 1g of CaCO3 and 20g of agar powder, and add deionized water to bring the volume to 1000ml. Autoclave the culture medium at 121℃ for 20 minutes.

[0101] The method for inducing zoospores is the same as in Example 2.

[0102] Comparative Example 4

[0103] Carrot culture medium preparation method: Cut 200g of fresh carrots into small pieces with sides of about 1cm, put them into 1000ml of deionized water and boil for 30 minutes. Filter through double-layer gauze to remove the residue. Add 20g of agar powder and add deionized water to make up to 1000ml. Autoclave the culture medium at 121℃ for 20 minutes.

[0104] The method for inducing zoospores is the same as in Example 2.

[0105] Comparative Example 5

[0106] Preparation of oat-sucrose medium: Add 30g of oat flakes to 1000mL of deionized water, incubate at 60℃ for 1 hour, and filter through double-layer gauze to remove residue. Add 20g of agar powder and bring the volume to 1000mL with deionized water. Autoclave the medium at 121℃ for 20 minutes.

[0107] The method for inducing zoospores is the same as in Example 2.

[0108] Comparative Example 6

[0109] Preparation method of orange juice culture medium: Peel fresh sweet oranges, cut them into pieces, and squeeze out the juice. Take 50ml of orange juice, add 20g of agar powder, and add deionized water to make up to 1000ml. Autoclave the culture medium at 121℃ for 20min.

[0110] The method for inducing zoospores is the same as in Example 2.

[0111] Comparative Example 7

[0112] Culture medium preparation method: Crush 200g of fresh, prickly cucumber in a food processor and filter through double-layered gauze. Crush 30g of fresh pepper leaves in the same way and collect the filtrate. Add 15g of agar powder to the filtrate and dilute with deionized water to 1L. Sterilize at 121℃ for 20min. When using, melt the culture medium and pour it into a sterile petri dish. After solidification, it becomes the culture medium plate.

[0113] The method for inducing zoospores is the same as in Example 2.

[0114] Comparative Example 8

[0115] Culture medium preparation method: Crush 200g of fresh, prickly cucumber in a food processor, filter through double-layered gauze, add 15g of agar powder to the filtrate, and bring the volume to 1L with deionized water. Sterilize at 121℃ for 20min. Dissolve 0.75g of vitamin C in 50mL of 1% oxalic acid solution, filter through a sterile filter with a pore size of 0.22μm, and store separately at 4℃ as an additive. When using, melt the culture medium and cool it to about 50℃, add 0.1% (by volume) of the vitamin C additive, mix well, pour into sterile petri dishes, and allow to solidify to form the culture medium plates.

[0116] The method for inducing zoospores is the same as in Example 2.

[0117] Comparative Example 9

[0118] Culture medium preparation method: Crush 30g of fresh pepper leaves and filtrate the liquid. Add 15g of agar powder to the filtrate, and dilute to 1L with deionized water. Sterilize at 121℃ for 20min. Dissolve 0.75g of vitamin C in 50mL of 1% oxalic acid solution, filter through a sterile filter with a 0.22μm pore size, and store separately at 4℃ as an additive. When using, melt the culture medium and cool it to about 50℃, add 0.1% (by volume) of the vitamin C additive, mix well, pour into sterile petri dishes, and allow to solidify to form the culture medium plates.

[0119] The method for inducing zoospores is the same as in Example 2.

[0120] Comparative Example 10

[0121] Culture medium preparation method: Crush 100g of fresh, prickly cucumber in a food processor and filter through double-layered gauze. Crush 30g of fresh pepper leaves in the same way and collect the filtrate. Add 15g of agar powder to the filtrate and dilute to 1L with deionized water. Sterilize at 121℃ for 20min. Dissolve 0.75g of vitamin C in 50mL of 1% oxalic acid solution, filter through a sterile filter with a 0.22μm pore size, and store separately at 4℃ as an additive. When using, melt the culture medium and cool to approximately 50℃. Add 0.1% (by volume) of the vitamin C additive, mix well, pour into sterile petri dishes, and allow to solidify to form the culture medium plates.

[0122] The method for inducing zoospores is the same as in Example 2.

[0123] Comparative Example 11

[0124] Culture medium preparation method: Crush 300g of fresh, prickly cucumber in a food processor and filter through double-layered gauze. Crush 30g of fresh pepper leaves in the same way and collect the filtrate. Add 15g of agar powder to the filtrate, and dilute to 1L with deionized water. Sterilize at 121℃ for 20min. Dissolve 0.75g of vitamin C in 50mL of 1% oxalic acid solution, filter through a sterile filter with a 0.22μm pore size, and store separately at 4℃ as an additive. When using, melt the culture medium and cool to approximately 50℃, add 0.1% (by volume) of the vitamin C additive, mix well, pour into sterile petri dishes, and allow to solidify to form the culture medium plates.

[0125] The method for inducing zoospores is the same as in Example 2.

[0126] Comparative Example 12

[0127] Culture medium preparation method: Crush 200g of fresh, prickly cucumber in a food processor and filter through double-layered gauze. Crush 10g of fresh pepper leaves in the same way and collect the filtrate. Add 15g of agar powder to the filtrate, and dilute to 1L with deionized water. Sterilize at 121℃ for 20min. Dissolve 0.75g of vitamin C in 50mL of 1% oxalic acid solution, filter through a sterile filter with a 0.22μm pore size, and store separately at 4℃ as an additive. When using, melt the culture medium and cool to approximately 50℃, add 0.1% (by volume) of the vitamin C additive, mix well, pour into sterile petri dishes, and allow to solidify to form the culture medium plates.

[0128] The method for inducing zoospores is the same as in Example 2.

[0129] Comparative Example 13

[0130] Culture medium preparation method: Crush 200g of fresh, prickly cucumber in a food processor and filter through double-layered gauze. Crush 40g of fresh pepper leaves in the same way and collect the filtrate. Add 15g of agar powder to the filtrate, and dilute to 1L with deionized water. Sterilize at 121℃ for 20min. Dissolve 0.75g of vitamin C in 50mL of 1% oxalic acid solution, filter through a sterile filter with a 0.22μm pore size, and store separately at 4℃ as an additive. When using, melt the culture medium and cool to approximately 50℃, add 0.1% (by volume) of the vitamin C additive, mix well, pour into sterile petri dishes, and allow to solidify to form the culture medium plates.

[0131] The method for inducing zoospores is the same as in Example 2.

[0132] Comparative Example 14

[0133] Culture medium preparation method: Crush 200g of fresh, prickly cucumber in a food processor and filter through double-layered gauze. Crush 10g of fresh pepper leaves in the same way and collect the filtrate. Add 15g of agar powder to the filtrate and dilute to 1L with deionized water. Sterilize at 121℃ for 20min. Dissolve 0.25g of vitamin C in 50mL of 1% oxalic acid solution, filter through a sterile filter with a 0.22μm pore size, and store separately at 4℃ as an additive. When using, melt the culture medium and cool to approximately 50℃, add 0.1% (by volume) of the vitamin C additive, mix well, pour into sterile petri dishes, and allow to solidify to form the culture medium plates.

[0134] The method for inducing zoospores is the same as in Example 2.

[0135] Comparative Example 15

[0136] Culture medium preparation method: Crush 200g of fresh, prickly cucumber in a food processor and filter through double-layered gauze. Crush 10g of fresh pepper leaves in the same way and collect the filtrate. Add 15g of agar powder to the filtrate and dilute to 1L with deionized water. Sterilize at 121℃ for 20min. Dissolve 1.50g of vitamin C in 50mL of 1% oxalic acid solution, filter through a sterile filter with a 0.22μm pore size, and store separately at 4℃ as an additive. When using, melt the culture medium and cool to approximately 50℃. Add 0.1% (by volume) of the vitamin C additive, mix well, pour into sterile petri dishes, and allow to solidify to form the culture medium plates.

[0137] The method for inducing zoospores is the same as in Example 2.

[0138] Example of effect

[0139] Each case was repeated at least three times. One-way, one-way factorial analysis was performed using IBM SPSS Statistics 22 software and Fisher's LSD (Least Significant Difference) method to analyze the significance of differences between cases at the P < 0.05 level (see Table 1). Table 1 shows that the sporulation induction effect of the culture medium of this invention is significantly better than that of other control cases, with the number of induced zoospores being more than 10 times that of the control cases.

[0140] Table 1 Comparative analysis of zoospore yield in each example and comparative example.

[0141]

[0142]

[0143] Note: The differences in tropical phytoalexinus among the culture media of each case were not significant. However, there were no significant differences among cases containing the same letter in the LSD one-way one-way analysis at the P < 0.05 level.

[0144] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Inducing tropical Phytophthora ( Phytophthora tropicalis The culture medium for producing zoospores is characterized by, Based on parts by weight, it comprises the following components: 150-250 servings of cucumber 20-30 parts by weight of pepper leaves Vitamin C 0.00001~0.00002 parts by weight; It also includes 15-20 parts by weight of agar powder; The method of using the cucumber is to crush the cucumber, filter it through double-layer gauze to remove the residue, and collect the filtrate. The method of using the pepper leaves is to crush the pepper leaves, filter them through double-layer gauze to remove the residue, and collect the filtrate. The method of using the vitamin C is to prepare a 1000-fold stock solution of the vitamin C and store it at 4°C. When using, add it to the culture medium solution cooled to 50°C.

2. The culture medium as described in claim 1, characterized in that, Based on parts by weight, it comprises the following components: 150 servings of cucumber 20 parts by weight of pepper leaves Vitamin C 0.00002 parts by weight or 200 servings of cucumber 30 parts by weight of pepper leaves Vitamin C 0.000015 parts by weight or 250 servings of cucumber 25 parts by weight of pepper leaves Vitamin C 0.00002 parts by weight or 200 servings of cucumber 30 parts by weight of pepper leaves Vitamin C 0.00001 parts by weight.

3. The method for preparing the culture medium as described in claim 1 or 2, characterized in that, The preparation method includes: taking 150-250g of the cucumber, crushing it, and then filtering it; Take 20-30g of the pepper leaves, crush them, collect the filtrate, add 15-20g of agar powder to the filtrate, add deionized water to make up to 1L, and sterilize at 121℃ for 20min; Dissolve 0.5-1g of the vitamin C in 50mL of 1% oxalic acid solution, filter, and refrigerate at 4°C to obtain the vitamin C additive. When using, melt the culture medium and cool it to 50°C, add 0.1% of the vitamin C additive, mix well, pour into a sterile petri dish, and solidify to obtain the culture medium plate.

4. The application of the culture medium as described in claim 1 or 2, or the culture medium prepared by the preparation method as described in claim 3, in inducing zoospores of Phytophthora tropicalis.

5. A method for inducing zoospores of *Phytophthora tropicalis*, characterized in that, Phytophthora tropicalis is inoculated into the culture medium as described in claim 1 or 2 or the culture medium prepared by the method described in claim 3, and cultured.

6. The induction method as described in claim 5, characterized in that, The steps include the following: Step 1: Inoculate the tropical phytophthora into the culture medium and incubate at 26°C for 4-7 days until the mycelium covers the entire plate; Step 2: Press the mycelium to adhere it to the culture medium, and induce it under light at 25~28℃ and 4000~6000lx for 24~48h; Step 3: After adding sterile water, refrigerate at 4℃ for 45~90 min, then keep at 26℃ for 30~60 min, and collect the spore suspension; Step 4: Observe and count the number of zoospores.

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